Anti-Inflammatory Effect of Ethanolic Extract from Myagropsis

Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 http://www.biomedcentral.com/1472-6882/12/171

RESEARCH ARTICLE Open Access Anti-inflammatory effect of ethanolic extract from Myagropsis myagroides on murine macrophages and mouse ear edema Eun-Ji Joung1, Min-Sup Lee1, Ji-Woong Choi1, Jong-Soon Kim1, Taisun Shin2, Bok-Mi Jung2, Na Young Yoon3, Chi-Won Lim3, Jae-Il Kim1 and Hyeung-Rak Kim1*

Abstract Background: This study aims to investigate anti-inflammatory effect of ethanolic extract of Myagropsis myagroides (EMM) in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and the phorbol 12-myristate 13-acetate (PMA)-induced ear edema in mice, and to clarify its underlying molecular mechanisms.

Methods: The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blotting. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunocytochemistry and reporter gene assay, respectively. PMA-induced mouse ear edema was used as the animal model of inflammation. Anti-inflammatory compounds in EMM were isolated using high-performance liquid chromatography and identified by nuclear magnetic resonance.

Results: EMM significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. EMM strongly suppressed nuclear translocation of NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. EMM reduced ear edema in PMA-induced mice. One of the anti-inflammatory compounds in EMM was identified as 6,6’-bieckol. Conclusions: These results suggest that the anti-inflammatory properties of EMM are associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines through the inhibition of NF-κB pathway in LPS-stimulated macrophages. Keywords: Anti-inflammation, Myagropsis myagroides, MAPK, NF-κB, RAW 264.7 cells

Background Activated macrophages play a pivotal role in inflamma- Inflammation is a complex response of host defense tory diseases by excessive production of pro- against microbial infection, endotoxin exposure, or cell inflammatory cytokines, including tumor necrosis factor injury, and ultimately leads to the restoration of normal (TNF)-α, interleukin (IL)-1β, and IL-6, and inflammatory cell structure and function. Macrophages are known to mediators such as nitric oxide (NO) and prostaglandin play a key role in host defense mechanism, and are acti- E2 (PGE2) [3,4]. Expressions of these cytokines and med- vated by exposure to interferon-γ, pro-inflammatory iators are regulated by the activation of nuclear factor- cytokines, and bacterial lipopolysaccharides (LPSs) [1,2]. kappaB (NF-κB), which is critically involved in the pathogenesis of rheumatism and other chronic inflammatory diseases [5]. In the unstimulated condition, * Correspondence: [email protected] 1Department of Food Science and Nutrition, Pukyong National University, NF-κB is located in the cytoplasm as an inactive complex Busan 608-737, South Korea bound to inhibitor of kappaB and (IκB)-α. Treatment of Full list of author information is available at the end of the article LPS activates the IκB-α kinase (IKK) complex, resulting

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in the phosphorylation, ubiquitination, and degradation USA). 4’,6-Diamidino-2-phenylindole (DAPI), Alexa W of IκB-α, inducing the translocation of NF-κB into the Fluor 488-conjugated secondary antibody, Lipofecta- nucleus for transcriptional activation. The activation of mine/Plus, and TRIzol were purchased from Invitro- NF-κB is also regulated by cellular kinases such as gen (Carlsbad, CA, USA). LPS (Escherichia coli O55: mitogen-activated protein kinases (MAPKs) and Akt B5), bovine serum albumin (BSA), 2',7'-dichlorofluores- [6,7]. Hence, substances which inhibit the activation of cein diacetate (DCF-DA), dimethyl sulfoxide (DMSO), NF-κB are considered as potential anti-inflammatory indomethacin (Indo), and phorbol 12-myristate 13- agents. acetate (PMA) were purchased from Sigma-Aldrich Myagropsis myagroides is one of the most abundant Corporation (St. Louis, MO, USA). Specific kinase inhibi- brown algae, which has been used as a part of the diet tors (Wortmannin, PD98059, SP600125, and SB203580) and folk medicines in Korea, Japan, and China for cen- were purchased from Abcam (Cambridge, UK). turies. This species contains high level of phlorotannins and pigments. Recently, phlorotannins have been Preparation of EMM reported to have several biological activities, such as M. myagroides was harvested from the coast of Busan, antioxidation [8], antidiabetic complications [9], antiam- South Korea, in April 2010. Taxonomic identification nesia [10], anti-inflammation [11], and hepatoprotection was confirmed by an agal taxonomist (C.K Choi), at the [12]. As a biological activity of M. myagroides, previ- Department of Ecological Engineering, Pukyong ous study revealed a cytoprotective effect on carbon National University, Korea. The seaweed was rinsed in tetrachloride-induced hepatotoxicity by its antioxidant tap water to remove salt, and air-dried under sunshine properties [13]. Recently, fucoxanthin isolated from for 3 days. A dried sample was ground with a hammer M. myagroides inhibited the production of inflammatory mill and the powder was stored at −20°C until used. The mediators and pro-inflammatory cytokines via inhibiting dried powder (500 g) of M. myagroides was extracted κ NF- B activation in RAW 264.7 cells [14]. However, we three times with 96% ethanol (3 L/each) for 3 h at 70°C. found an anti-inflammatory activity in the ethanolic ex- The combined extracts were filtered and concentrated tract of sun-dried M. myagroides (EMM) in LPS- using rotary vacuum evaporator (Tokyo Eyela, Tokyo, stimulated RAW 264.7 cells. Since fucoxanthin prone to Japan) at 40°C and freeze-dried EMM (89.7 g) was rapidly oxidized during sun-drying due to instability under stored in freezer (−70°C) until used. oxygen and sun light exposure, we hypothesized that other anti-inflammatory compounds might be exist in EMM. Furthermore, we confirmed that EMM does not contain Cell culture and viability assay any detectable amount of fucoxanthin by HPLC chroma- Murine macrophage RAW 264.7 cells (ATCC, Rockville, tographic analysis at 410 nm. To our knowledge, no previ- MD, USA) were cultured at 37°C in DMEM supplemen- ous study has been reported on the anti-inflammatory ted with 10% FBS, penicillin (100 units/mL), and μ activity of M. myagroides except fucoxanthin. Therefore, streptomycin sulfate (100 g/mL). Cell viability was we investigated the anti-inflammatory and anti-edema determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) properties of EMM and its underlying mechanisms ® using cultured RAW 264.7 cells and identified anti- assay using a CellTiter96 AQueous One Solution Cell ’ inflammatory compound in EMM. Proliferation assay kit according to the manufacturer s in- struction. Briefly, cells were inoculated at a density of 5 × 5 Methods 10 cells/well into 96-well plate and cultured at 37°C for μ Chemicals 24 h. The culture medium was replaced with 200 Lof ® CellTiter96 AQ One Solution Cell Proliferation serial dilutions of EMM, and the cells incubated for 24 ueous μ assay kit, luciferase assay kit, murine NF-κB promoter/ h. The medium was replaced with 95 L fresh medium μ luciferase DNA, pRL-TK DNA and reverse transcript- and 5 L MTS solution. After 1 h, the absorbance at 490 ase were obtained from Promega (Madison, WI, nm was measured using a microplate reader (Glomax USA). Primary antibodies and secondary antibodies Multi Detection System, Promega). were purchased from Santa Cruz Biotechnology (Santa

Cruz, CA, USA). Enhanced chemiluminescence (ECL) Measurement of NO, PGE2, TNF-α, IL-1α and IL-6 detection kit was obtained from GE Healthcare Bio- RAW 264.7 cells were plated in a 12-well plate at a Science (Piscataway, NJ, USA). Dulbecco’s modified density of 1 × 106 cells/well and incubated for 24 h. Cul- Eagle's medium (DMEM), penicillin-streptomycin mix- tured cells were treated with various concentrations of ture, 0.25% trypsin-ethylenediaminetetraacetic acid EMM for 1 h, and then stimulated with 1 μg/mL LPS (EDTA), and fetal bovine serum (FBS) were purchased for 24 h. Cultured media were collected after centrifuga- from Gibco BRL Life Technologies (Grand Island, NY, tion at 2,000g for 10 min and stored at −70°C until Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 3 of 11 http://www.biomedcentral.com/1472-6882/12/171

tested. The nitrite concentration in the cultured media mM EDTA, 1 mM DTT, and 0.5 mM phenylmethylsul- was measured as an indicator of NO production, accord- fonyl fluoride (PMSF), pH 7.9] and incubated on ice for ing to the Griess reaction [15]. Levels of PGE2, IL-1β, 15 min. After vortexing for 10 s, homogenates were IL-6, and TNF-α in cultured media were quantitated by separated into supernatants (cytoplasmic compartments) enzyme-linked immunosorbent assay (ELISA, R&D Sys- and pellets (nuclear components) by centrifugation at tems, Minneapolis, MN, USA) according to the manu- 13,000g for 10 min. The pellet was gently resuspended facturer’s instructions. in 40 μL complete lysis buffer (50 mM HEPES/KOH, 50 mM KCl, 1 mM DTT, 300 mM NaCl, 1% IGEPAL CA- Reverse transcription-polymerase chain reaction (RT-PCR) 630, 0.1 mM EDTA, 10% glycerol, and 0.5 mM PMSF, RAW 264.7 cells placed in a 6-well plate were pretreated pH 7.9) and centrifuged at 13,000g for 20 min at 4°C. with EMM for 1 h and then stimulated with LPS for 6 h. The supernatant was used as the nuclear extract. Total RNA from each group was isolated with the TRI- zol reagent. Five microgram of total RNA was used for Western immunoblot analysis reverse transcription using oligo-dT-adaptor primer and RAW 264.7 cells were incubated with various concentra- superscript reverse transcriptase. PCR was carried out tions of EMM for 1 h and stimulated with LPS (1 μg/mL) with the gene-specific primers: COX-2 sense, 5’- for 30 min. RAW 264.7 cells were washed twice with cold CAGCAAATCCTTGCTGTTCC-3’; COX-2 antisense, PBS and lysed with lysis buffer (50 mM Tris–HCl, pH 7.5, 5’-TGGGCAAAGAATGCAAACAT-3’;iNOSsense,5’- 150 mM NaCl, 1% IGEPAL CA-630, 1% Tween 20, 0.1% CACCTTGGAGTTCACCCAGT-3’;iNOSantisense, SDS, 1 mM Na3VO4,10μg/mL leupeptin, 50 mM NaF, 5’-ACCACTCGTACTTGGGATGC-3’; glyceraldehyde and 1 mM PMSF) on ice for 1 h. After centrifugation at 3-phosphate dehydrogenase (GAPDH) sense, 5’- 18,000g for 10 min, the protein concentrations in the GACCCCTTCATTGACCTCAA-3’; GAPDH antisense, supernatants were determined, and aliquots of the protein 5’-CTTCTCCATGGTGGTGAAGA-3’.GAPDHwas (40 μg) were separated by sodium dodecyl sulfate- used as an internal standard to evaluate relative ex- polyacrylamide gel electrophoresis (SDS-PAGE) and pression of COX-2 and iNOS. Densitometric analysis transferred onto a nitrocellulose membrane. The mem- ofthedataobtainedfromat least three independent brane was blocked with 5% nonfat dry milk in Tris- experiments was performed using cooled CCD camera buffered saline with 0.1% Tween 20 (TBST) for 1 h, fol- system EZ-Capture II and CS analyzer ver. 3.00 soft- lowed by the incubation for 2 h with primary antibody in ware (ATTO & Rise Co., Tokyo, Japan). TBST containing 5% nonfat dry milk. The blots were treated with horseradish peroxidase-conjugated secondary Transient transfection and luciferase assay antibody in TBST containing 5% nonfat dry milk for 1 h, Murine NF-κB promoter/luciferase DNA (1 μg) along and immune complexes were detected using an ECL de- with 20 ng control pRL-TK DNA was transiently trans- tection kit. Densitometric analysis of the data obtained fected into 2 × 105 RAW 264.7 cells/well in a 24-well from at least three independent experiments was per- plate using Lipofectamine/Plus reagents for 40 h. Cells formed using cooled CCD camera system EZ-Capture II pretreated with 0–100 μg/mL EMM for 1 h were stimu- and CS analyzer ver. 3.00 software. lated with LPS (1 μg/mL) for 6 h. Each well was washed twice with cold phosphate-buffered saline (PBS), har- Immunofluorescence analysis vested in 100 μL of lysis buffer (0.5 mM HEPES, pH 7.8, RAW 264.7 cells were maintained on glass coverslips 1% Triton N-101, 1 mM CaCl2, and 1 mM MgCl2) and (SPL Lifesciences Co., Gyeonggi-do, Korea) to analyze used for the measurement of luciferase activity using a nuclear localization of NF-κB in 24-well plates for 24 h. luciferase assay kit. Luminescence was measured on a Cells treated with EMM for 1 h were incubated with TopCount microplate scintillation and luminescence LPS (1 μg/mL) for 30 min as described in Lee et al. [16]. counter (PerkinElmer, Santa Clara, CA, USA) in single- Cells were fixed in 4.0% paraformaldehyde in PBS for 15 photon counting mode for 0.1 min/well, following a 5 min at room temperature, and then permeabilized with min adaptation in the dark. Luciferase activity was nor- 0.5% Triton X-100 in PBS for 10 min. Cells were washed malized to the expression of control pRL-TK. with PBS and blocked with 3% BSA/PBS for 30 min. Thereafter, cells were incubated with an anti-NF-κB Preparation of cytosolic and nuclear extracts polyclonal antibody diluted in 3% BSA/PBS for 2 h, and W RAW 264.7 cells (5 × 106 cells/well ) pretreated with incubated with Alexa Fluor 488-conjugated secondary EMM for 1 h were stimulated with LPS for 0.5 h. Cells antibody diluted in 3% BSA/PBS for 1 h. Cells were were washed twice with cold PBS and harvested. Cell stained with 2 μg/mL DAPI and images were captured pellets were resuspended in 300 μL of hypotonic buffer using an LSM700 laser scanning confocal microscope [10 mM HEPES/KOH, 10 mM KCl, 2 mM MgCl2, 0.1 (Carl Zeiss, Oberkochen, Germany). Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 4 of 11 http://www.biomedcentral.com/1472-6882/12/171

Mouse model and PMA-induced ear edema (treatment). Inhibition percentage (IP) was expressed as Animal studies were conducted after the experimental a reduction in weight compared to the PMA-treated protocols and procedures were approved by the Animal group. Ethics Committee of the Pukyong National University. ICR mice (male, 25–30 g) were purchased from the Isolation of phlorotannins from EMM Samtako Bio Korea Co. (Gyeonggido, Korea) and permit- Aliquots of EMM were separated by Shimadzu high- ted free access to a standard chow diet and tap water. performance liquid chromatography (HPLC) system with All mice were acclimatized for 1 week prior to the Luna RP-18 [Luna C18(2), 5 μm, 250 × 10 mm, Phe- experiments and maintained at 22 ± 2°C with a relative nomenex, Torrence, CA, USA]. The separation of EMM humidity of 50 ± 5% and 12 h light–dark cycle. Ear was conducted using the mobile phase of 0.1% formic edema was induced on the right ear of mouse with PMA acid in water (solvent A) and 0.1% formic acid in aceto- according to Garrido et al. [16]. Briefly, the control nitrile (solvent B). The elution profile consisted of a lin- group received normal saline, and the other three groups ear gradient from 20 to 100% solvent B for 110 min and include a PMA alone, PMA + Indo (a positive control), hold for 15 min and then re-equilibration of the column and EMM administered groups (PMA + EMM). The left with 20% solvent B for 15 min. The flow rate was 0.34 ear (reference) received the vehicle (30 μL acetone). mL/min at 35°C oven temperature and detection was PMA (6 μg/ear in 30 μL acetone) was applied to the performed at 270 nm. The purity was determined by inner surface of the right ear of ICR mouse. EMM (90 HPLC (purity: >99%) and the chemical structure of the μg per ear in 30 μL acetone) or Indo (1 mg/ear in 30 μL isolated compounds were identified as eckol [17] and acetone) was topically administered 1 h before PMA ap- 6,6’-bieckol [8,12] by comparing their 1H- and 13C-NMR plication. Six hours after PMA application, mice were data. killed by cervical dislocation and a 6 mm diameter disc from each ear was removed with a metal punch and Statistical analysis weighed. Ear edema weight was calculated by subtract- Data were expressed as the means ± standard deviations ing the weight of the left ear from the right ear (SDs) from at least three separate experiments unless

(A) (B) 35 5000 a a 30 b 4000 a a 25 b 3000 20 b

15 b 2000 concentration (pg/mL) concentration

10 2 1000 Nitrite concentration (M) 5 b PGE 0 0 LPS (1 µg/mL) -+++++ LPS (1 µg/mL) -++++- EMM (µg/mL) - - 10 25 50 100 EMM (µg/mL) - - 25 50 100 100 (C) 120

100

Relative cell viability (%) viability cell Relative 20

0 LPS (1 µg/mL) -++++ EMM (µg/mL) -- 25 50 100

Figure 1 Effect of EMM on LPS-induced NO and PGE2 production in RAW 264.7 cells. Cells pretreated with different concentrations of EMM for 1 h were stimulated with LPS (1 μg/mL) for 24 h. The treated culture media were used to measure the amount of NO production (A) and

PGE2 production (B). Cytotoxic effect of EMM was measured by MTS assay (C). Values are the means ± SDs of three independent experiments. ap < 0.05 indicates significant differences compared to the control group. bp < 0.05 indicates significant differences compared to the LPS-only treated group. Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 5 of 11 http://www.biomedcentral.com/1472-6882/12/171

otherwise indicated. Data were analyzed using one-way mediated inhibition of NO and PGE2 production in analysis of variance. Differences were considered signifi- LPS-stimulated macrophages is associated with downre- cant at values of p < 0.05. All analyses were performed gulation of iNOS and COX-2 expression at transcrip- using SPSS for Windows, version 10.07 (SPSS Inc., tional level. Chicago, IL, USA). Effect of EMM on LPS-induced pro-inflammatory Results cytokines

Effect of EMM on LPS-induced NO and PGE2 production Increased levels of IL-1β (Figure 3A), IL-6 (Figure 3B), NO production, measured as nitrite, was increased by and TNF-α (Figure 3C) in RAW 264.7 cells by LPS LPS treatment; however, EMM significantly reduced NO stimulation were dramatically reduced in a dose- levels in LPS-stimulated cells in a dose-dependent man- dependent manner by exposure to EMM (p < 0.05). This ner (p < 0.05, Figure 1A). Increased PGE2 production by result indicates that EMM efficiently suppressed LPS- LPS was also significantly suppressed by 100 μg/mL induced IL-1β, IL-6, and TNF-α release, which supports EMM in RAW 264.7 cells (Figure 1B). To exclude the the hypothesis that EMM inhibits the initial phase of the possibility that the decreased NO and PGE2 levels were LPS-stimulated inflammatory response. due to cell death, cytotoxicity of EMM was determined by MTS assay. The result demonstrated that EMM showed Effect of EMM on LPS-induced activation and no cytotoxicity in RAW 264.7 cells up to 100 μg/mL translocation of NF-κB (Figure 1C). Thus, the inhibitory effects of EMM on NO Confocal microscopy revealed that NF-κB/p65 was dis- and PGE2 production were not due to cytotoxicity. tributed mostly in the cytoplasm in unstimulated cells. After stimulation with LPS, most cytoplasmic NF-κB/p65 Effect of EMM on LPS-induced iNOS and COX-2 proteins were translocated into the nucleus, as shown by strong and mRNA expression NF-κB/p65 intensity in the nucleus (Figure 4A). The level As shown in Figure 2, EMM strongly inhibited iNOS of p65 in the nucleus was markedly reduced by treatment protein production in a dose-dependent manner; how- with EMM. Considering the inhibitory effects of EMM ever, COX-2 protein production was only inhibited by on LPS-induced NF-κB translocation into nucleus, we 100 μg/mL EMM, which is similar pattern observed in determined the effect of EMM on the promoter activity the PGE2 secretion (Figure 1B). In addition to iNOS pro- of NF-κB in LPS-stimulated cells. Data suggested that tein, EMM also inhibited iNOS mRNA expression in a EMM treatment significantly inhibited LPS-induced dose-dependent manner. Like inhibition of COX-2 pro- NF-κB promoter activity (Figure 4B, p <0.05). tein, COX-2 mRNA expression was partially inhibited by LPS treatment resulted in increased IKKβ phosphoryl- 100 μg/mL EMM. These results suggest that the EMM- ation and IκB-α degradation compared to non-treated

Figure 2 Effect of EMM on LPS-induced iNOS and COX-2 protein and mRNA expression in RAW 264.7 cells. (A) Cells were pretreated with indicated concentration of EMM for 1 h and stimulated with LPS (1 μg/mL) for 16 h. Forty microgram of proteins were subjected to 10% SDS-PAGE. The expression of iNOS, COX-2, and β-actin protein was detected by Western blotting using corresponding antibodies. (B) Cells were pretreated with EMM for 1 h and stimulated with LPS for 6 h, and then total RNA was prepared for RT-PCR. The results presented are representative of three independent experiments. ap < 0.05 indicates significant differences compared to the control group. bp < 0.05 indicates significant differences compared to the LPS-only treated group. http://www.biomedcentral.com/1472-6882/12/171 Joung LS.Dt r en D ftreidpnetexperiments. independent three of SDs ± means are Data ELISA. 8-ojgtdscnayatbd.Tence eesandwt AIadteiae eecpue ycnoa irsoy ( microscopy. confocal by captured were h. images 6 the for and stimulation DAPI with NF- stained with were NF- transfected nuclei min. The 30 antibody. for secondary stimulation 488-conjugated LPS by followed h 1 4 Figure ocnrtoso M o ,adte tmltdwt P o 4h IL-1 h. 24 for LPS with stimulated then b and h, 1 for EMM of concentrations 3 Figure oteLSol rae group. treated LPS-only the to EMM (100 p .5idctssgiiatdfeecscmae oteLSol rae group. treated LPS-only the to compared differences significant indicates 0.05 < LPS (1 ta.BCCmlmnayadAtraieMedicine Alternative and Complementary BMC al. et NF- feto M natvto n rnlcto fNF- of translocation and activation on EMM of Effect fet fEMo r-nlmaoyctkn rdcin nLSsiuae A 6. cells. 264.7 RAW LPS-stimulated in productions cytokine pro-inflammatory on EMM of Effects (A) Merged µ DAPI µ B/p65 g/mL) g/mL) a LPS (1 EMM ( κ EMM ( LPS (1 p rmtrcnann uieaeDAwr rtetdwt aiu ocnrtoso M o olwdb LPS by followed h 1 for EMM of concentrations various with pretreated were DNA luciferase promoter-containing B .5idctssgiiatdfeecscmae otecnrlgroup. control the to compared differences significant indicates 0.05 < (C) (A) µ µ µ α µ β - + - TNF- concentration (ng/mL) IL-1 concentration (pg/mL) g/mL) g/mL) g/mL) g/mL) 100 150 250 300 200 150 250 100 200 50 50 0 0 - 25 50 - + + + + 100 - 100 - 25 50 - + + - 100 100 - + a a κ b a /6 uuiswr rbdb anti-NF- by probed were subunits B/p65 + b b 2012, b b 12 a p κ 11Pg f11 of 6 Page :171 .5idctssgiiatdfeecscmae otecnrlgroup. control the to compared differences significant indicates 0.05 < nRW247cells. 264.7 RAW in B β LPS (1 EMM ( EMM ( LPS (1 ( A (B) ,I- ( IL-6 ), µ µ

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NF- B promoter activity (AU) 0 B 10 25 15 20 30 ,adTNF- and ), 0 5 κ oylnlpiayatbd n lx Fluor Alexa and antibody primary polyclonal B ( - + + + + - - 25 50 100 100 A el eeperae ihadwtotEMfor EMM without and with pretreated were Cells ) b p .5idctssgiiatdfeecscompared differences significant indicates 0.05 < a α - + - + + + - 25 50 100 100 ( C nteclue ei eemaue by measured were media cultured the in ) b a b el eetetdwt various with treated were Cells b b b b B Cells ) W Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 7 of 11 http://www.biomedcentral.com/1472-6882/12/171

Figure 5 Inhibitory effect of EMM on the degradation of IκB-α and the activation of NF-κB in LPS-stimulated RAW 264.7 cells. Cells were incubated with various concentrations of EMM for 1 h, and then stimulated with LPS (1 μg/mL) for 30 min. Cytosolic and nuclear fractions were prepared and analyzed by Western blotting using corresponding antibodies. The results presented are representative of three independent experiments. ap < 0.05 indicates significant differences compared to the control group. bp < 0.05 indicates significant differences compared to the LPS-only treated group. control group, and EMM treatment suppressed IKKβ phos- (SP600125), or p38 inhibitor (SB203580). As shown in phorylation and IκB-α degradation, recovered the control Figure 6B, IκB-α phosphorylation was remarkably sup- level of cytosolic IκB-α in a dose-dependent manner pressed by p38 inhibitor, which is consistent with the (Figure 5). As a result of IκB-α degradation, the increased result of Figure 6A. Other inhibitors such as Wortmannin, NF-κB level in nucleus after LPS stimulation was reduced PD98059, and SP600125, moderately inhibited the phos- by EMM treatment in a dose-dependent manner (Figure 5). phorylation of IκB-α. These data confirm additional However, phosphorylation of c-Jun, a component of tran- characteristics of EMM on the phosphorylation of scription factor AP-1, was not reduced by EMM treatment. MAPKsandAktproteinsforNF-κB activation in re- These results indicate that the EMM-mediated inhibition sponse to LPS. of iNOS, COX-2, and pro-inflammatory cytokines production was mainly regulated by the transcription factor Effect of EMM on PMA-induced ear edema in mouse NF-κB in LPS-stimulated macrophages. Ear edema was induced by the application of PMA to the mouse ears. As shown in Figure 7, the application of PMA Effect of EMM on LPS-induced phosphorylation of MAPKs for 6 h significantly increased the weight of ear edema. Indo, and Akt a common clinical non-steroidal anti-inflammatory drug, As shown in Figure 6A, EMM inhibited phosphorylation of was used as positive control for the inhibitory effect on the JNK, p38 MAPK, ERK, and Akt induced by LPS in RAW ear edema. As shown in Figure 7, pre-treatment of 1 mg/ear 264.7 cells, suggesting the additional characteristics of of Indo could effectively reduce ear edema after 6 h PMA EMM to regulate NF-κB pathway via blocking the phos- stimulation (p < 0.05). Similarly, pre-administration of phorylation of MAPKs and Akt proteins in response to LPS EMM (90 μg/ear) markedly inhibited the PMA-induced signal. To further confirm the association of these signaling ear edema with 66.8% inhibition (p <0.05). molecules with the EMM’s anti-inflammatory effect, IκB-α phosphorylations in LPS-stimulated RAW 264.7 cells were Isolation of anti-inflammatory compounds from EMM measuredinthepresenceofPI3K/Aktpathwayinhibitor EMM was further separated by reverse-phase column (Wortmannin), ERK inhibitor (PD98059), JNK inhibitor chromatography (Figure 8A) for the isolation of anti- Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 8 of 11 http://www.biomedcentral.com/1472-6882/12/171

Figure 6 Effect of EMM on the phosphorylations of MAPKs and Akt in RAW 264.7 cells. (A) Cells pretreated with indicated concentrations of EMM for 1 h were stimulated with LPS (1 μg/mL) for 30 min. (B) Cells pretreated with indicated concentrations of EMM or inhibitors for 1 h were stimulated with LPS (1 μg/mL) for 30 min. Whole cell lysates (40 μg) were analyzed by Western blotting using corresponding antibodies. The results presented are representative of three independent experiments. ap < 0.05 indicates significant differences compared to the control group. bp < 0.05 indicates significant differences compared to the LPS-only treated group. inflammatory compound(s). The chemical structures of the myagroides, from the comparison of their NMR spectra isolated compounds were identified as eckol and 6,6’- with the published spectral data. We obtained 2.5 mg of bieckol, which were isolated for the first time from M. eckol and 5.6 mg of 6,6’-bieckol from 1 g of EMM. The purified 6,6’-bieckol significantly inhibited LPS-induced iNOS and COX-2 production in a dose-dependent manner (p < 0.05, Figure 8B), however, eckol did not affect on the production of inflammatory proteins. Furthermore, pre- administration of 6,6'-bieckol (30 μg/ear) markedly inhibited the PMA-induced ear edema with 64.1% inhibition (Figure 7, p < 0.05). The result indicates that 6,6’-bieckol is one of the anti-inflammatory compounds in EMM.

Discussion The present study was undertaken to examine the anti- inflammatory effect of EMM on LPS-stimulated murine macrophage cells. To further understood the molecular mechanisms of EMM, we investigated the effects of EMM on the secretion of NO, PGE2,TNF-α,IL-1β, and IL-6, the expression of iNOS and COX-2, and the activation of NF-κB. Our results indicated that EMM effect- Figure 7 Effect of EMM on ear edema induced by PMA in α β ’ ively inhibited the secretion of NO, PGE2, TNF- ,IL-1, mouse. EMM, 6,6 -bieckol or indomethacin (Indo) was administered κ topically on inner surface of the right ears of mice 1 h before and IL-6 through a blockade of the NF- BandMAPK application of PMA. Six hours after application of PMA, ear edema pathways in LPS-stimulated macrophages. The inhibitory and the inhibition percentage (IP) of the ear edema by the effect of EMM on the expression of inflammatory media- treatment were calculated. ap < 0.05 indicates significant differences tors suggested one of the mechanisms responsible for its b compared to the non-treated group. p < 0.05 indicates significant anti-inflammatory action and its potential for use as a differences compared to the PMA-only treated group. therapeutic agent for treating inflammatory diseases. Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 9 of 11 http://www.biomedcentral.com/1472-6882/12/171

Figure 8 Identification of eckol and 6,6’-bieckol in EMM. (A) HPLC chromatogram of EMM and the chemical structures of eckol and 6,6’- bieckol (insert). (B) Cells pretreated with different concentrations of eckol or 6,6’-bieckol for 1 h were stimulated with LPS for 16 h. The expression levels of iNOS and COX-2 in cells were analyzed by Western blotting using corresponding antibodies. Relative density ratios of each protein over β-actin are shown below the blots. The results presented are representative of three independent experiments. ap < 0.05 indicates significant differences compared to the control group. bp < 0.05 indicates significant differences compared to the LPS-only treated group.

Under pathological conditions, excessive inflammatory the inhibition of inflammatory response. Our results indi- mediators and pro-inflammatory cytokines produced by cate that the inhibition of NO and PGE2 production by activated macrophages cause inflammatory process and EMM in LPS-stimulated macrophage cells is associated act synergistically with other inflammatory mediators with downregulation of iNOS and COX-2 genes (Figure 2), [2,4,18]. Compounds able to reduce NO or PGE2 produc- which seems the first addressing transcriptional inhibition tion may be attractive as anti-inflammatory agents and, for of iNOS and COX-2 by EMM. this reason, the inhibitory effects of natural compounds NF-κB is a transcription factor actively involved in the on NO or PGE2 productions have been rigorously studied transcriptional induction of iNOS and COX-2 gene [1,3]. to develop therapeutic agent against inflammatory diseases Like NO and PGE2 produced by iNOS and COX-2, respect- [17,19,20]. Also, excessive production of pro-inflammatory ively, the release of pro-inflammatory cytokines is regulated cytokines plays a critical role in acute inflammatory by NF-κB pathway and plays an important role in the in- responses as well as chronic inflammatory diseases. Recent duction of the innate immune response of the acquired im- studies have shown that in vivo or in vitro treatments of mune response [23]. It has been well studied that the natural compounds are effective in reducing inflammation inhibition of NF-κB activation by black tea extract is asso- by the suppression of pro-inflammatory cytokines, which ciated with the phosphorylation, ubiquitination, and subse- may ameliorate inflammation-related diseases, including quent degradation of IκB via ubiquitin-proteosome pathway atherosclerosis, cancer, and inflammatory arthritis [21,22]. [24]. Although biochemical actions of EMM on NF-κBreg- Thus, the regulation of those molecules is important to ulations remain unknown, the present study showed that Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 10 of 11 http://www.biomedcentral.com/1472-6882/12/171

EMM potentially inhibits the proteolytic degradation of found in brown seaweeds, have strong antioxidant [18,27] IκB-α and the NF-κB promoter-driven luciferase expression and anti-inflammatory [11,28] activities. The multifunc- induced by LPS in RAW 264.7 cells. Therefore, these tional antioxidant activity of phlorotannins is highly asso- results demonstrate the ability of EMM to inhibit NF-κB ciated with phenol rings which act as electron traps to activation in response to LPS signal in RAW 264.7 cells. scavenge peroxy, superoxide-anions, and hydroxyl radicals Hence it is likely that reductions of iNOS, COX-2, and pro- [29]. Specific mechanisms of phlorotannins on the anti- inflammatory cytokine expression in RAW 264.7 cells are inflammatory actions are not clearly defined. Considering largely associated with activation/deactivation of NF-κB cellular signaling of polyphenols, phlorotannins may not pathway. This report is, to our best knowledge, the novel merely exert their effects as free radical scavenger, but findings to address the functions of EMM through NF-κB may also modulate inflammatory cellular signaling pro- pathway in response to LPS treatment. teins, including NF-κB and AP-1 transcription factors NF-κB is also regulated by various signaling kinases in- [30]. Recent studies demonstrated that phlorofucofur- cluding MAPKs (ERK, JNK, and p38) and Akt, which are oeckol A and 6,6’-bieckol isolated from Ecklonia spp. alle- groups of signaling molecules to play key roles in NF-κB viate inflammatory response to LPS by inhibiting NF-κB activation [2,3]. MAPKs have been suggested to be pathway with its intrinsic antioxidant activity [11,28]. In involved in pro-inflammatory signaling cascades and in the present study, the isolated 6,6’-bieckol from EMM the activation of NF-κB in LPS-stimulated immune cells remarkably inhibited the production of iNOS and [2,11]. Therefore, anti-inflammatory mechanisms are COX-2 proteins, indicating 6,6’-bieckol is one of the closely related with inhibition of MAPKs in activated principle anti-inflammatory compounds in EMM. RAW 264.7 cells. In this study, we found that phosphorylation of MAPKs in response to LPS was inhibited by Conclusions EMM treatment (Figure 6A). Interesting finding of this We demonstrated that EMM inhibited the secretion of study is that the activation of Akt, a downstream regulator inflammatory mediators, such as NO and PGE2, and of PI3K, was also inhibited by EMM in response to LPS pro-inflammatory cytokines, including TNF-α, IL-1β, signal in RAW 264.7 cells. Precise mechanisms by which and IL-6, in LPS-stimulated RAW 264.7 macrophages. EMM modulate this PI3K/Akt pathway in the LPS stimu- Moreover, the inhibitory effect of EMM was associated lation remains unclear. Considering a recent study with a with inactivation of the NF-κB pathway via blocking the phlorofucofuroeckol A, which indicated a link between phosphorylation of MAPKs and Akt. In addition, the ROS and PI3K/Akt pathway in regulations of inflamma- results of in vivo study showed that EMM can be applic- tory genes [7,11], it is speculated that the antioxidant ac- able to a topical anti-inflammatory agent. tivity of EMM or unknown components in EMM may be related to inhibition of Akt phosphorylation. Thus, it is Abbreviations COX-2: Cyclooxygenase 2; DAPI: 4’,6-Diamino-2-phenylindole; likely that inhibition of MAPKs and Akt phosphorylation DMEM: Dulbecco’s modified Eagle’s medium; DMSO: Dimethyl sulfoxide; by EMM may contribute to the EMM-mediated inhibition EMM: Ethanolic extract of Myagropsis myagroides; ERK: Extracellular signal of NF-κB pathway in LPS-stimulated RAW 264.7 cells. regulated kinase; ELISA: Enzyme-linked immunosorbent assay; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HPLC: High- Topical application of PMA to mouse skin induces in- performance liquid chromatography; IκB: Inhibitor of κB; IKK: IkappaB kinase; flammatory response and this is a well-established in vivo IL: Interleukin; iNOS: Inducible nitric oxide synthase; JNK: c-Jun N-terminal model for the evaluation of various anti-inflammatory kinase; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinase; NMR: Nuclear magnetic resonance; NF-κB: Nuclear factor-κB; NO: Nitric oxide; agents. PMA can activate a wide variety of cell types that PBS: Phosphate-buffered saline; PGE2: Prostaglandin E2; may contribute to acute inflammation, resulting in an in- PMSF: Phenylmethylsulfonyl fluoride; PCR: Polymerase chain reaction; crease in epidermal tissue swelling and infiltration of in- TNF-α: Tumor necrosis factor-α; PMA: Phorbol 12-myristate 13-acetate. flammatory cells [25,26]. The results from our in vivo μ Competing interests experiments clearly showed that EMM (90 g/ear) sup- The authors declare that they have no competing interests. pressed 66.8% of PMA-induced skin swelling. Further- more, 6,6’-bieckol (30 μg/ear) isolated firstly from M. Authors’ contributions E-JJ carried out the main experiment and wrote the manuscript. M-SL myagroides in this study inhibited 64.1% of PMA-induced prepared EMM and performed partial Western blotting. J-WC performed skin swelling, indicating that it might be a main anti- immunocytochemistry. JSK performed ELISA for measuring the inflammatory compound in EMM. Although the inhibi- proinflammatory cytokines. TS and B-MJ isolated and identified anti- ’ inflammatory compounds from EMM. NYY and C-WL harvested M. tory activity of EMM is about one third of 6,6 -bieckol, myogroides. J-IK contributed to discuss and write the manuscript. H-RK EMM showed higher anti-edema activity than Indo com- designed and organized this study. All authors read and approved the final pared to its concentration. This result demonstrates that version of the manuscript. EMM can be used as a topical anti-inflammatory agent. Acknowledgements Like other polyphenolic compounds, phlorotannins, a This work was financially supported by the National Fisheries Research and class of compounds polymerized with phloroglucinol units Development Institute (RP-2012-FS-015) and Brain Busan 21. Joung et al. BMC Complementary and Alternative Medicine 2012, 12:171 Page 11 of 11 http://www.biomedcentral.com/1472-6882/12/171

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Kim H, Lee HS, Chang KT, Ko TH, Baek KJ, Kwon NS: Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by • Convenient online submission preventing activation of nuclear factor-κB. J Immunol 1995, 154:4741–4748. • Thorough peer review 16. Lee JY, Lee MS, Choi HJ, Choi JW, Shin T, Woo HC, Kim JI, Kim HR: Hexane • No space constraints or color ﬁgure charges fraction from Laminaria japonica exerts anti-inflammatory effects on lipopolysaccharide-stimulated RAW 264.7 macrophages via inhibiting • Immediate publication on acceptance NF-kappaB pathway. Eur J Nutr 2012, [Epub ahead of print]. • Inclusion in PubMed, CAS, Scopus and Google Scholar 17. Kim AR, Shin TS, Lee MS, Park JY, Park KE, Yoon NY, Kim JS, Choi JS, Jang BC, • Research which is freely available for redistribution Byun DS, Park NK, Kim HR: Isolation and identification of phlorotannins from Ecklonia stolonifera with antioxidant and anti-inflammatory properties. 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